ISSN 2278- 4136

ZDB-Number: 2668735-5

IC Journal No: 8192
Volume1Issue4

OnlineAvailableatwww.phytojournal.com

Journal of Pharmacognosy and Phytochemistry

Vol.1No.42012www.phytojournal.comPage|112

Formulation and Evaluation of an Herbal Anti-Inflammatory
Gel Containing Eupatorium Leaves Extract

Arvind Negi
1
*, Nimisha Sharma
1
, Mamta F. Singh
2
1. Guru Ram Das Post Graduate Institute of Management and Technology, Department of Pharmacy,
Rajpur Road, Dehradun, Uttarakhand, India.
[E-mail: arvind_n2007@yahoo.co.in ]
2. Sardar Bhagwan Singh (PGI) of Biomedical Sciences, Balawala, Dehradun, Uttarakhand, India.


This study evaluated a noble herbal gel formulation containing extract from the leaves of Eupatorium adenophorum
for its topical anti-inflammatory activity against carrageenan induced oedema. Gelling agent used in this study was
1% w/w concentration of carbopol- 934. The studies were conducted on Albino Wistar rats of either sex (150-200
gm). Change in oedema volume of the rat hind paw was measured. The anti-inflammatory effect produced after
topical administration of herbal gel formulation on Carrageenan-induced hind paw oedema exhibited a high degree
of reproducibility. The initial physicochemical parameters of formulations i.e. pH, viscosity, spreadability,
extrudability and stability were also examined. The pH of all the formulations was near about 6.8, which lies in the
normal pH range of the skin. The preparation was stable under normal storage conditions and did not produce any
skin irritation, i.e., erythema and oedema for about a month, when applied over the skin.
Keyword: Herbal Gel, Inflammation, Eupatorium Leaves extract, Oedema.


1. Introduction: Eupatorium (Asteraceae) is a
large genus of herbs, shrubs or under shrubs,
distributed chiefly in tropical America, a few
species occurring in Europe, Africa, Asia and
India
[1]
. Different parts of the species Eupatorium
adenophorum Spreng are used in Ayurveda and
other folk medicines for the treatment of cut,
wounds, as antifungal
[2]
, antimicrobial
[3]
, and
analgesic agent
[4]
.
A recent study on the ethanolic leaf extract of E.
adenophorum has reported anti-inflammatory
activity in dinitrofluorobenzene induced foot paw
oedema in mouse for the first time
[5]
. Since,
inflammation is a pathophysiological response of
living tissues to injuries that leads to the local
accumulation of plasmatic fluid and blood cells
1

and if not controlled, it can lead to development
of diseases such as chronic asthma, rheumatoid
arthritis, multiple sclerosis, inflammatory bowel
disease, and so forth
[6-11]
, the present study was
designed to formulate and evaluate the herbal gel
formulation containing methanol extract of E.
adenophorum leaves for its anti-inflammatory
potential in carrageenan induced paw oedema in
rats.

2. Material and Methods
2.1 Preparation of methanolic extract
The leaves of E. adenophorum Spreng were
collected from Rajpur Road, Dehradun (Uttara
khand) and authenticated by Botanical Survey of
India, Northern Regional Centre, Dehradun with
Journal of Pharmacognosy and Phytochemistry
Vol.1No.42012www.phytojournal.comPage|113

the Accession number 1127802, 1127803. The

leaves were dried under shade and then powdered
coarsely with a mechanical grinder. The powder
was passed through sieve No. 40 and stored in an
airtight container for further use. Hundred grams
of powdered leaves were extracted with methanol
as a solvent by hot extraction method using
soxhlet apparatus. The resulting extract was
cooled and filtered. The filtrate was evaporated in
vacuum to give a residue.

2.2 Formulation of topical preparation
Herbal gel was prepared using carbopol-934 as a
gelling agent in 1% w/w concentration with
deionized water using mechanical stirrer. The pH
of the gel was adjusted to neutral by addition of
small quantities of triethanolamine with
continuous stirring. 1 % w/w herbal extract of E.
adenophorum was added to the gel and stirred for
sufficient time for homogeneous mixing of
extract in gel base. Prepared gel was filled in
collapsible tubes and stored at a cool and dry
place. Physical parameters such as colour,
appearance, and feeling on application were
recorded. pH of the gel was recorded using a pH
meter.

2.3 Viscosity
Viscosity of gel was measured by using
Brookfield viscometer with spindle #7.

2.4 Extrudability
[12]

The gel formulations were filled in standard
capped collapsible aluminum tubes and sealed by
crimping to the end. The weights of the tubes
were recorded. The tubes were placed between
two glass slides and were clamped. 500 gm was
placed over the slides and then the cap was
removed. The amount of the extruded gel was
collected and weighed. The percent of the
extruded gel was calculated (>90% extrudability:
excellent, >80% extrudability: good, >70%
extrudability: fair).

2.5 Spreadability
[13]

Spreadability was determined by the apparatus
which consists of a wooden block, which was
provided by a pulley at one end. By this method
spreadability was measured on the basis of slip
and drag characteristics of gels. An excess of gel
(about 2 g) under study was placed on the ground
slide. The gel was then sandwiched between this
slide and another glass slide having the
dimension of fixed ground slide and provided
with a hook. A 1 kg weight was placed at the top
of the two slides for 5 minutes to expel air and to
provide a uniform film of the gel between the
slides. Excess of the gel was scrapped off from
the edges. The top plate was then subjected to
pull of 80 g with the help of string attached to the
hook and the time (in seconds) required by the
top slide to cover a distance of 7.5 cm was noted.
A shorter interval indicated better spreadability.
Spreadability was calculated using the following
formula:

S = M L / T

Where,
S = Spreadability
M = Weight in the pan (tied to the upper slide)
L = Length moved by the glass slide
T = Time (in sec.) taken to separate the upper slide from
the ground slide.

2.6 Stability study
[14]

The stability study was performed as per ICH
guidelines. The formulated gel was filled in
collapsible tubes and stored at different
temperatures and humidity conditions, viz. 252
C / 605% RH, 302 C / 655% RH, 402 C /
755% RH for a period of three months and
studied for appearance, pH and spreadability.

2.7 Primary Dermal Irritation Index (PDII)
Dermal irritation is the production of reversible
damage to the skin following the application of a
test substance for up to 4 hours. Primary dermal
irritation index (PDII) is a method for classifying
topical formulations into various categories based
on acute toxic reactions observed upon single
application of a formulation on skin. Based on the
PDII score, the formulation can be graded as
irritating or non-irritating.

2.8 Selection and maintenance of animals
Healthy young male albino rabbits, weighing 1.5
2 kg at the start of the experiment, were used as
Journal of Pharmacognosy and Phytochemistry
Vol.1No.42012www.phytojournal.comPage|114

the experimental animals in the present study

(Clinical Ethical clearance No:
1145/a/07/CPCSEA/2011/6). The animals were
housed together in a clean tank which was
spacious enough for free movement of the
animals and accommodation to hold drinking
water and feed. Room temperature was 25030
C, humidity was 45-55% with a light period of
12 h (06.00 to 18.00). The animals were fed with
commercially available standard pellet chow and
filtered tap water.

2.9 Preparation of animals
Approximately 24 hours before the test, fur was
removed by closely clipping the dorsal area of the
trunk of the animals. Care was taken to avoid
abrading the skin and only animals with healthy,
intact skin were used for the study.

2.10 Application of the herbal gel
Half a gram of the herbal gel, as the test
substance, was applied to an area of
approximately 6 cm
2
of skin and covered with a
gauze patch. The patch was loosely held in
contact with the skin by means of a suitable semi-
occlusive dressing for 4 hours and was then
removed. At the end of the exposure period, i.e. 4
hours, residual test substance was removed
without altering the existing response or the
integrity of the epidermis. Observations were
recorded an hour after the removal of the patch.
Control animals were prepared in the same
manner and 0.5 gram of the gel base, i.e. gel
formulated using all the ingredients except the
herbal mixture, was applied to the control animals
and observations were made similar to the test
animals. Both the control and the test animals
were observed every day for any occurrence of
skin irritation or toxic reactions such as oedema
or erythema. Per observation of skin, a value
between 0 and 4 was recorded where 0 meant no
skin erythema and eschar formation and 1, 2, 3
and 4 stood for very slight, well defined,
moderate and severe erythema to eschar
formation, respectively. It also scored from 04,
where 0 stood for no oedema and 4 stood for
severe oedema.


Primary Dermal Irritation Index (PDII) =

PDIIobservedon12 24 48 72hrs
4


2.11 Classification system based on PDII
< 0.5: non-irritating, 0.5-2.0: slightly irritating,
2.1-5.0: moderately irritating and >5.0: severely
irritating.

2.12. 28 days repeated dose dermal toxicity of
the developed herbal gel formulation
28 days repeated dose dermal toxicity of the
herbal gel formulation was conducted on the
rabbit skin to evaluate the cumulative toxicity of
the herbal gel upon repeated application along
with the study of behavioral, hematological and
biochemical parameters. Both the control and the
test animals were observed every day for any
occurrence of toxic or irritation reactions such as
oedema or erythema.

2.13 Hematological analysis
Blood samples of all the test and control rabbits
were collected by vein puncture on the 14
th
and
on the 28
th
day of the study. Estimation of
haemoglobin percentage was done using
haemocytometer.

2.14 Biochemical analysis
For determining blood sugar, total cholesterol,
creatinine, urea, total and direct bilirubin, protein,
SGOT, SGPT, alkaline phosphatase and acid
phosphatase, blood samples were collected
separately from each control and experimental
animal by retro orbital puncture on the 14
th
and
28
th
day of the study.

2.15 Evaluation of anti-inflammatory activity
Animals
Albino Wistar rats of either sex, weighing 150
200 g were used. They were housed in standard
environmental conditions and fed with standard
rodent diet with water ad libitum. All animal
procedures were followed in three groups
(Control, Test and Standard) of six animals each.

Journal of Pharmacognosy and Phytochemistry
Vol.1No.42012www.phytojournal.comPage|115

2.16 Carrageenan-induced rat paw

oedema
[15,16]

Animals were fasted for 24 hrs before the
experiment with water ad libitum. Approximately
50l of a 1% suspension of carrageenan in saline
was prepared 1 h before each experiment and was
injected into the plantar side of right hind paw of
rat. 0.2 g of herbal gel containing 1% E.
adenophorum extract was applied to the plantar
surface of the hind paw by gentle rubbing 50
times with the index finger. Rats of the control
groups received the plain gel base and 0.2 g 1%
Valdecoxib gel applied in the same way was used
as a standard. Drugs or placebo were applied 1h
before the carrageenan injection. Paw volume
was measured immediately after carrageenan
injection and at 1, 2, 3 and 4 hrs intervals after
the administration of the noxious agent by using a
plethysmometer.

2.17 Statistical Analysis
Data are reported as the meanSEM (Standard
Error Mean) and were analyzed statistically by
means of analysis of variance (ANOVA)
followed by Students t-test. Values of p<0.05 are
regarded as significant.

3. Results
The herbal gel was prepared and subjected to
evaluation of various parameters. The gel was
greenish in colour with a translucent appearance
and cooling sensation throughout the evaluation
period. The pH was constant throughout the study
to about 6.8 and the gel did not produce any
irritation upon application to the skin.
Extrudability was excellent while spreadability
was less variant after performing stability studies
from that of the initially prepared gel. The initial
viscosities were recorded at 25 C. Furthermore,
the stability studys results revealed the
preparation was stable under normal storage
conditions.

The Primary Dermal Irritation Index (PDII) of the
formulation was 0.00; hence, according to OECD
guidelines the formulation can be classified as
non-irritant to the rabbit skin. No clinical signs of
dermal toxicity were observed in any of the
animals treated with the test substance upon
repeated application of the herbal gel for up to 28
days.

The control and the experimental rabbits showed
no signs of convulsions. Hematological profiles
of the experimental rabbits were studied after the
repeated application of herbal gel daily for 28
days. Hematological parameters such as total
counts of RBC and WBC, differential count of
WBC and hemoglobin percentage were normal
before treatment and after 14 and 28 days of
application of the herbal gel. Biochemical
parameters of blood such as blood sugar, total
cholesterol, creatinine, urea, total and direct
bilirubin, total protein, SGOT, SGPT, alkaline
phosphatase and acid phosphatase of both test
and the control rabbits were evaluated for any
change in these parameters due to the application
of the herbal gel with respect to the control
rabbits. The changes were statistically
insignificant.

These results indicated that the herbal gel had no
adverse effects on the biochemical parameters of
the blood. The anti-inflammatory effect produced
after topical administration of herbal gel
formulation on Carrageenan-induced hind paw
oedema exhibited a high degree of
reproducibility.

Table 1: Extrudability of the herbal gel at the time of
preparation (Mean SEM)

Extrudability Mean of three tubes (Initial
month)
Net wt of formulation in
tube (g)
12.340.011
Wt. of gel extruded (g) 11.320.014
Extrudability amount
percentage
91.730.005




Journal of Pharmacognosy and Phytochemistry
Vol.1No.42012www.phytojournal.comPage|116

Table 2: Spreadability of the herbal gel during the evaluation period (Mean SEM)


Evaluation Condition
Spreadability (g.cm/sec)
Mean of three readings
Initial month 30.030.015
After 3 months at 252 C/ 605% RH 30.010.012
After 3 months at 302 C/ 655% RH 29.980.012
After 3 months at 402 C/ 755% RH 29.920.014

Table 3: Viscosity of the herbal gel at the time of preparation

RPM Viscosity (cps) Initial month
50 22910
75 18790
100 15260
150 13089

Table 4: Effect of topical administration of Herbal gel on carrageenan-induced rat paw oedema

Early Phase Percentage Inhibition Late Phase Percentage Inhibition
Control 53.92.051 - 94.21.933 -
Standard 39.22.342 27.27 61.50.987 34.71
Herbal gel 49.41.120 8.35 75.70.851 19.64

4. Conclusion
Carrageenan induced oedema is a biphasic
response. The first phase is mediated through the
release of histamine, serotonin and kinins
whereas the second phase is related to the release
of prostaglandin and slow reacting substances
[15]
.
The data presented in this study demonstrate that
E. adenophorum leaves extract in the form of gel
possess significant topical anti-inflammatory
properties, supporting their traditional use for the
treatment.

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